Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins

ABSTRACT

Immunoassays for malondialdehyde-modified low density lipoprotein (MDA-modified LDL) and oxidized low density lipoprotein (OxLDL), monoclonal antibodies (and the cell lines for them) for use in the assays, and a storage-stable standard (which may be used as a calibrator and/or control) are disclosed. MDA-modified LDL and OxLDL are implicated in atherosclerosis and its etiology.

BACKGROUND

The present invention relates to assays, antibodies (particularlymonoclonal antibodies), and standards for detection (i.e., determinationof the presence and/or quantitation of the amount) of oxidized lowdensity lipoprotein (OxLDL) and malondialdehyde-modified low densitylipoprotein (MDA-modified LDL) in samples, the samples typically beingderived from body fluids or tissues.

Lipoproteins are multicomponent complexes of protein and lipids. Eachtype of lipoprotein has a characteristic molecular weight, size,chemical composition, density, and physical role. The protein and lipidare held together by noncovalent forces.

Lipoproteins can be classified on the basis of their density asdetermined by ultracentrifugation. Thus, four classes of lipoproteinscan be distinguished: High Density Lipoproteins (HDL), IntermediateDensity Lipoproteins (IDL), Low Density Lipoproteins (LDL), and Very LowDensity Lipoproteins (VLDL).

The purified protein components of a lipoprotein particle are calledapolipoproteins (apo). Each type of lipoprotein has a characteristicapolipoprotein composition. In LDL the prominent apolipoprotein proteinis apo B-100. Apo B-100 is one of the longest single chain polypeptidesknown and consists of 4536 amino acids. Of these amino acids the lysineresidues or moieties (there are 356 such lysine residues or moieties)can be substituted or modified by aldehydes (e.g., malondialdehyde).

Oxidation of the lipids in LDL (whether in vitro, e.g., bycopper-induced oxidation, or whether in vivo) results in the generationof reactive aldehydes, which can then interact with the lysine residuesor moieties of apo B-100. The outcome of this lysine substitution ormodification is that the resulting OxLDL, which is also MDA-modifiedLDL, is no longer recognized by the LDL receptor at the surface offibroblasts but by scavenger receptors at the surface of macrophages. Atleast 60 out of the 356 lysines (or lysine residues or moieties) of apoB-100 have to be substituted in order to be recognized by the scavengerreceptors (see document number 1 of the documents listed near the end ofthis application, all of which documents are hereby incorporated intheir entireties for all purposes). The uptake of such OxLDL bymacrophages results in foam cell generation, which is considered to bean initial step in atherosclerosis.

Endothelial cells under oxidative stress (e.g., in acute myocardialinfarction patients) and activated blood platelets also producealdehydes, which interact with the lysine moieties in apo B-100,resulting in the generation of aldehyde-modified LDL that is alsorecognized by the scavenger receptors. However, the lipids in thisaldehyde-modified LDL are not oxidized. Enzymatic activity inmacrophages (e.g. myeloperoxidase) results in the oxidation of both thelipid and the protein moieties of LDL. All these pathways result inaldehyde-type modification of the protein moiety of LDL.

In vitro experiments and experiments in animal models have suggestedthat OxLDL and/or aldehyde-modified LDL may contribute to theprogression of atherosclerosis by inducing endothelial dysfunction, foamcell generation, smooth muscle cell proliferation, and plateletactivation (for review see document number 2). A positive correlationbetween the levels of autoimmune antibodies that cross-react withaldehyde-modified LDL and the progression of carotid atheroscleroticlesions in patients suggested that OxLDL and/or aldehyde-modified LDLmight contribute to the progression of human atherosclerosis (seedocument 3).

However, the possibility that the autoimmune antibodies were directedagainst other aldehyde-modified proteins, e.g., albumin, could not beexcluded. Therefore, the contribution of OxLDL and aldehyde-modified LDL(whether or not resulting from oxidation of the lipid moiety) to humanatherosclerosis may be able to be established when non-invasive teststhat are specific for these substances (i.e., have high affinity forthose substances in preference to other substances) become available.

Because the underlying mechanisms of oxidation of LDL may be differentin different patient populations (e.g., in diabetes patients, chronicrenal failure patients, heart transplant patients) and because at leastsome of the mechanisms may be independent of lipid oxidation, such testsshould be specific for both OxLDL and aldehyde-modified LDL (e.g.,MDA-modified LDL) and thus preferentially be based on the detection ofconformational changes that specifically occur in the apo B-100 moietyof LDL following aldehyde-type substitution of lysine residues. In otherwords, there is a need for such non-invasive tests (i.e., assays) thatare highly specific for the analytes of interest (i.e., MDA-modified LDLand OxLDL). There is also a need for antibodies that are specific forthe analytes of interest. There is also a need for a stable standard(e.g., to be used as calibrator and/or control) for the assays.

SUMMARY OF THE INVENTION

An invention satisfying those needs and having other features andadvantages that will be apparent to those skilled in the art has nowbeen developed. The present invention provides antibody-based assaysthat are capable of specifically quantitating (quantifying) both OxLDLand aldehyde-modified LDL or MDA-modified LDL in samples, e.g., samplesderived from body fluids (like plasma or serum) or tissues. The presentinvention also provides monoclonal antibodies useful in those assays andcell lines (hybridomas) that produce those antibodies. The presentinvention also provides a storage-stable standard, which can be used asa calibrator and as a control for the assays. Having such a standard isnecessary for having reliable and reproducible and therefore usefulassays.

Broadly, in one aspect the present invention concerns an immunologicalassay for the detection and/or quantification of MDA-modified LDL andOxLDL in a sample, said assay comprising:

a) contacting the sample with a first antibody that has high affinityfor MDA-modified LDL and OxLDL; and

b) thereafter visualizing and/or quantifying a binding reaction betweenthe first antibody and the MDA-modified LDL and OxLDL present in thesample;

wherein the MDA-modified LDL and OxLDL for which the first antibody hashigh affinity contain at least 60 substituted lysine moieties per apoB-100 moiety.

That assay may, for example, be a competitive assay, a sandwich assay,an immunohistochemical assay, etc. “Competitive assays” are well-knownand any competitive assay may be used in this invention provided it iswithin the limitations of the invention and that the benefits of theinvention can be achieved. “Sandwich assays” are well-known and anysandwich assay may be used in this invention provided it is within thelimitations of the invention and that the benefits of the invention canbe achieved. “Immunohistochemical assays” are well-known and anyimmunohistochemical assay may be used in this invention provided it iswithin the limitations of the invention and that the benefits of theinvention can be achieved.

In another aspect, the present invention concerns an immunologicalsandwich assay for the detection and/or quantification of MDA-modifiedLDL in a sample in which assay a first antibody that has a high affinityfor MDA-modified LDL is bound to a substrate, said assay comprising:

(a) contacting the sample with the substrate having bound to it thefirst antibody under binding conditions so that at least some of anyMDA-modified LDL in the sample will bind to the first antibody;

(b) thereafter removing unbound sample from the substrate;

(c) thereafter contacting the substrate with a second antibody that hasa high affinity for MDA-modified LDL; and

(d) thereafter visualizing and/or quantifying the MDA-modified LDL thatwas present in the sample;

wherein the MDA-modified LDL for which the first antibody and the secondantibody have high affinity contains at least 60 substituted lysinemoieties per apo B-100 moiety.

As used herein (including the claims), “high affinity” means an affinityconstant (association constant) of at least about 5×10⁸ M⁻¹, desirablyat least about 1×10⁹ M⁻¹, preferably at least about 1×10¹⁰ M⁻¹, and mostpreferably of at least about 1×10¹¹M⁻¹. As used herein (including theclaims), “low affinity” means an affinity constant (associationconstant) of less than about 1×10⁷ M⁻¹, desirably less than about1×10⁶M⁻¹, and preferably less than about 1×10⁵ M⁻¹. Affinity constantsare determined in accordance with the appropriate method described inHolvoet et al. (4).

The antibodies that can be used in this invention will bind withMDA-modified LDL and/or OxLDL whose apo B-100 moieties contain at least60, desirably at least about 90, more desirably at least about 120,preferably at least about 180, more preferably at least about 210, andmost preferably at least about 240 substituted lysine residues per apoB-100 moiety. The range of lysine substitution will generally be from 60to about 240 and preferably from about 120 to about 240 substitutedlysine moieties per apo B-100 moiety.

Each new monoclonal antibody is highly specific for a conformationalepitope that is present when at least about 60, preferably at leastabout 120 lysine residues, are substituted and by virtue thereof candistinguish various markers or indications related to atherosclerosis.Antibodies recognizing epitopes present when less than about 60 lysinesare substituted or modified are less specific but are still useful(e.g., they may be used as the secondary antibody in a sandwich ELISA).

The preferred antibodies used herein are monoclonal antibodies mAb-4E6,mAb-1h11, and mAb-8A2. Their affinity constants for native LDL,MDA-modified LDL, and OxLDL are as follows: Antibody Native LDLMDA-modified LDL OxLDL mAb-4E6 less than 3 × 10¹⁰ 2 × 10¹⁰ 1 × 10⁶mAb-1H11 less than 3 × 10¹⁰ less than 1 × 10⁶ 1 × 10⁶ mAb-8A2 5 × 10⁹ 1× 10¹⁰ 1 × 10¹⁰

In yet another aspect, the present invention concerns (a) monoclonalantibody mAb-4E6 produced by hybridoma Hyb4E6 deposited at the BCCMunder deposit accession number LMBP 1660 CB on or about Apr. 24, 1997,(b) monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2 depositedat the BCCM under deposit accession number LMBP 1661 CB on or about Apr.24, 1997, (c) hybridoma Hyb4E6 deposited at the BCCM under depositaccession number LMBP 1660 CB on or about Apr. 24, 1997, and (d)hybridoma Hyb8A2 deposited at the BCCM under deposit accession numberLMBP 1661 CB on or about Apr. 24, 1997.

The antibodies used in the assays of this invention are preferably thosetwo (i.e., mAb-4E6 and mAb-8A2) as well as mAb-1H11. The cell line forantibody mAb-1H11 is produced by hybridoma Hyb1H11, which was depositedat the BCCM under deposit accession number LMBP 1659 CB on or about Apr.24, 1997.

The BCCM is the Belgian Coordinated Collections of Microorganismsauthorized by the “Budapest Treaty of 28 Apr. 1977 on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.” Its address is c/o The University of Gent, K.Ledeganckstraat 35, B-9000 Gent, Belgium.

The assay may be of a type that is well-known, such as an Enzyme-LinkedImmunosorbent Assay (ELISA). For example, in the case of a sandwichELISA, mAb-4E6 (for MDA-modified LDL and OxLDL) or mAb-1H11 (forMDA-modified LDL) may be bound to a solid substrate and subsequentlycontacted with a sample to be assayed. After removal of the sample,binding between the specific antibody and OxLDL and/or MDA-modified LDLcaptured out of the sample can be visualized and/or quantified bydetection means. Detection means may be a labeled, less specificsecondary antibody that recognizes a different part of the apo B-100moiety of the captured analyte (e.g., mAb-8A2).

In the case of a competitive ELISA, a solid substrate coated with OxLDLor MDA-modified LDL may be contacted for a predetermined period of timewith the monoclonal antibody mAb-4E6 and a sample thought or known tocontain OxLDL and/or MDA-modified LDL, after which period of timeunbound antibody and sample are removed and a binding reaction betweenantibody and OxLDL and/or MDA-modified LDL bound to the substrate isvisualized and/or quantified. Quantification in a competitive ELISA isindirect because the binding between the antibody and the analyte in thesample is not measured but instead the amount of antibody that binds tothe known amount of OxLDL or MDA-modified LDL that is coated on (boundto) the substrate is measured. The more antibody bound to the knownamount of OxLDL or MDA-modified LDL coated on the substrate, the lessanalyte there was in the sample.

In yet another aspect, the present invention concerns a stable standardcontaining MDA-modified LDL whose extent of substitution of its lysinemoieties will remain essentially constant, over normal periods of timeduring normal storage for biological materials, the DA-modified LDL ofsaid standard being made by contacting (incubating) malondialdehyde withLDL at a predetermined molar ratio of malondialdehyde to the apo B-100moiety of the LDL.

“Over normal periods of time during normal storage for biologicalmaterials” as used herein refers to the time periods and conditionsunder which biological materials to be used in assays and otherlaboratory work are typically stored. Those conditions will typicallyinclude low temperature and in appropriate cases freezing, either withor without lyophilization. Depending on the particular biologicalmaterial, if the material is stored under the appropriate temperatureand other conditions (e.g., lack of vibration or other movement, properhumidity), the material may be stable for at least three months,desirably for over a year, preferably for over two years, and mostpreferably for over three years.

The standard preferably contains an agent that reduces the ability ofany metal ions present to catalyze oxidation of the LDL (e.g., achelating agent, such as EDTA) and/or one or more anti-oxidants (e.g.,BHT and/or Vitamin E). Preferably both the agent that reduces theability of any metal ions present to catalyze oxidation of the LDL andthe anti-oxidant are used. It has surprisingly been found that whenusing an antibody that is specific for both OxLDL and MDA-modified LDL,the storage-stable standard of this invention (containing MDA-modifiedLDL and not OxLDL) can be used. That eliminates the need to try toformulate, store, and use a stable standard containing OxLDL. OxLDL maycontinue to oxidize under typical storage conditions, making using as astandard a composition containing OxLDL difficult if not almostimpossible. EDTA will typically be used in concentrations of 0.5 to 5mM, preferably in concentrations of 0.5 to 2 mM. BHT will typically beused in concentrations of 5 to 50 μM, preferably in concentrations of 10to 20 μM. Vitamin E will typically be used in concentrations of 5 to 50μM, preferably in concentrations of 10 to 20 μM. The standard may alsocontain anti-platelet agents and coagulation inhibitors.

It has been found that LDL that has been modified by treatment with MDAis highly stable. Such MDA-modified LDL (which is not oxidized, i.e.,its lipid moiety is not oxidized) could be added to reference plasmasamples and those samples could be frozen and thawed without increasingthe extent of lysine substitution. Because the total number of lysineresidues in all apo B-100 molecules is identical, a constant MDA/apoB-100 molar ratio in the reaction mixture will result in an identicalnumber of substituted lysines in the MDA-modified LDL. In contrast, forexample, metal-ion mediated oxidation of LDL ultimately results in avariable extent of lysine substitution because it depends on theoxidation sensitivity of the LDL preparation, which by itself depends onfatty acid composition and antioxidant content, which are highlyvariable even in healthy control individuals.

As described below, a correlation between the oxidation of LDL and theextent of post-transplant atherosclerosis in heart transplant patientswas established using this invention. The relationship betweenendothelial injury and the modification of LDL was established inchronic renal failure patients that are at high risk for atheroscleroticcardiovascular disease. It was also demonstrated that endothelial injuryis an initial step in atherosclerosis.

Based on the characteristics of the oxidatively modified LDL from theplasma of heart transplant and chronic renal failure patients, it wasconcluded that cell-mediated aldehyde modification independent of lipidoxidation was at least partially involved. This finding furthersupported the hypothesis that an assay for oxidatively modified LDL hasto detect both OxLDL and aldehyde-modified LDL.

In yet another aspect, the invention concerns a kit for conducting asandwich assay for the determination of OxLDL or MDA-modified LDL orboth in a sample, said kit comprising a substrate on which is bound afirst antibody that has high affinity for OxLDL or MDA-modified LDL orboth, the OxLDL and MDA-modified LDL each having at least 60 substitutedlysine moieties per apo B-100 moiety, and a labeled antibody having ahigh affinity for OxLDL that becomes bound to the first antibody duringthe assay or for MDA-modified LDL that becomes bound to the firstantibody during the assay or for both that become bound to the firstantibody during the assay. Preferably the kit further comprises areactive substance for reaction with the labeled antibody (e.g., anenzyme) to give an indication of the presence of the labeled antibody.Preferably the kit also comprises the stable standards, e.g., in theform of stable calibrators and/or stable controls. Thus, e.g., the boundantibody may be mAb-4E6 or mAb-1H11 and the labeled antibody may bemAb-8A2.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are provided to help further describe theinvention, which drawings are as follows:

FIG. 1 illustrates the correlation between amounts of oxidized andMDA-modified LDL in coronary lesions in Watanabe heritablehyperlipidemic rabbits (A) and in miniature pigs (B).

FIG. 2 illustrates the accumulation of OxLDL and MDA-modified LDL incoronary arteries of cardiac explants of ischemic heart disease but notof dilated cardiomyopathy patients.

FIG. 3 illustrates the inhibition of the binding of mAb-4E6 toimmobilized OxLDL by native LDL, OxLDL and MDA-modified LDL in solution.

FIG. 4 illustrates a typical standard curve obtained with MDA-modifiedLDL in sandwich ELISA.

FIG. 5 illustrates levels of OxLDL and aldehyde-modified LDL inposttransplant plasma samples of heart transplant patients withdifferent extents of angiographically assessed coronary artery stenosis.

FIG. 6 illustrates the correlation between plasma levels of OxLDL andaldehyde-modified LDL and titers of specific autoantibodies.

FIG. 7 illustrates the correlation between plasma levels of OxLDL andaldehyde-modified LDL and of von Willebrand factor antigen.

These drawings are provided for illustrative purposes only and shouldnot be used to unduly limit the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be further described in conjunction with thefollowing examples, which are for illustrative purposes and which shouldnot be used to unduly limit the invention.

EXAMPLES Example 1

Preparation and Characterization of Antibodies Specific for OxLDL andfor Aldehyde-Modified LDL

Balb/c mice were immunized by intravenous and intraperitoneal injectionof either OxLDL or MDA-modified LDL. OxLDL was obtained by in vitroincubation of LDL (final apo B-100 concentration 700 μg/ml) with copperchloride (final concentration 640 μM) for 16 h at 37° C. MDA-modifiedLDL was prepared by incubation of LDL (final apo B-100 concentration 700μg/ml) with a 0.25 M MDA-solution for 3 h at 37° C. The numbers ofsubstituted lysines, measured in the TBARS assay, was typically 210 perapo B-100 molecule for OxLDL and 240 for MDA-modified LDL. Hybridomaswere obtained by PEG induced fusion of spleen lymphocytes derived fromimmunized mice with P3-X63/Ag-6.5.3 myeloma cells according to standardtechniques (4). The screening for hybridomas producing specificantibodies was performed with ELISA using microtiter plates coated withmalondialdehyde-modified LDL or copper-oxidized LDL. 308 hybridomas wereobtained after immunization of mice with either OxLDL (211) orMDA-modified LDL (97). Hyb4E6 produced antibodies specific for bothmalondialdehyde-modified and copper-oxidized LDL (mAb-4E6), and Hyb1H11produced antibodies specific for malondialdehyde-modified LDL (mAb-1H11)alone. The IgG fraction of the antibodies was purified by affinitychromatography on protein A-Sepharose and the affinity of the purifiedIgGs was determined in a solid phase radioimmunoassay and/or in ELISA.The K_(a) values of the monoclonal antibody mAb-4E6 were <10⁶ M⁻¹ fornative LDL and >10⁹ M⁻¹ for malondialdehyde-modified LDL andcopper-oxidized LDL. The K values for the monoclonal antibody mAb-1H11were <10⁶ M⁻¹ for both LDL and OxLDL and >10⁹ M⁻¹ formalondialdehyde-modified LDL. The K_(a) values for the monoclonalantibody mAb-8A2, obtained after immunization of mice with LDL, were>10⁹ M⁻¹ for all LDL forms. Delipidation of MDA-modified LDL and OxLDLresulted in a loss of the immunoreactivity of mAb-4E6, suggesting thatit is directed against a conformational epitope in the protein moiety ofoxidatively modified LDL.

Example 2

Use of mAb-4E6 for the Quantitation of OxLDL and MDA-Modified LDL inCoronary Lesions of Watanabe Heritable Hyperlipidemic Rabbits andMiniature Pigs on a Cholesterol Rich Diet

Coronary arteries were obtained from 2 and 5 month old Watanabeheritable hyperlipidemic rabbits (n=30) on normal chow or from miniaturepigs (n=26) which were fed a diet enriched in cholesterol (4%),saturated fat (14% beef tallow) and bile extract (1%) for 6 to 24 weeks.

Arterial specimens were submerged within 30 min after removal in PBS (pH7.4) containing 4% sucrose, 20 μM vitamin E and 10 μM butylatedhydroxytoluene as antioxidants, and 1 mM EDTA, snap-frozen in liquidnitrogen and stored at −80° C. Frozen 7 μM sections were stained withhematoxylin and eosin and with oil red 0 or immunostained as describedbelow. Morphometric parameters of atherosclerotic lesions were measuredby planimetry using the Leica 2 Quantimet color image analyzer(Cambridge, UK). The area within the external elastic lamina, theinternal clastic lamina and the lumen were measured. Media was definedas the area between the internal and external clastic lamina. Intima wasdefined as the area within the internal elastic lamina not occupied byvessel lumen.

Oxidized apo B-100 containing lipoproteins were detected with thespecific monoclonal antibody mAb-4E6, alkaline-phosphatase conjugatedrabbit-anti-mouse IgG antibodies and the fuchsin alkaline phosphatesubstrate system (Dako, Carpinteria, Calif.), and the absorbance wasmeasured in the color image analyzer. Specificity of immunostaining wasconfirmed by inhibition of staining with excess of copper-oxidized LDLbut not with native LDL or with malondialdehyde-modified albumin. Thestaining co-localized with that monoclonal antibody mAb-13F6, specificfor apo B-100. Absorbance (approximately 10%) measured with excesscopper-oxidized LDL was presumed to represent background staining.

FIG. 1 illustrates the correlation between the levels of oxidized apoB-100 containing lipoproteins, i.e. OxLDL and MDA-modified LDL, in thelesions and the mean intimal area of coronary lesions in Watanabehyperlipidemic rabbits (A) and in miniature pigs (B). Those data thusdemonstrate a correlation between the accumulation of OxLDL andMDA-modified LDL and the progression of coronary atherosclerotic lesionsin 2 different animal models. In Watanabe rabbits the progression of thelesions is due to the increase of LDL cholesterol associated with theheritable LDL receptor deficiency, whereas the progression in miniaturepigs is due to a diet-induced increase in LDL cholesterol.

Example 3

Immunohistochemistry

1. Introduction

This example is a typical example of the use of the highly specificantibody mAb-4E6 in immunohistochemistry applied to humanatherosclerotic lesions. In a similar manner corresponding experimentsmay be performed, for which certain conditions can be adapted by theskilled person using his common knowledge in the field.

2. Material and Methods

Coronary artery specimens, obtained at the time of transplantation frompatients with ischemic heart disease (n=7) or dilated cardiomyopathy(n=7), were treated as described earlier (document 7). The specimenswere collected within 30 min after removal of the heart in PBS (pH 7.4)containing 4% sucrose, 20 μM vitamin E and 10 μM butylatedhydroxytoluene as antioxidants, and 1 mM EDTA, and were stored at −80°C. Frozen 7 μm thick sections were cut and stained with hematoxylin andeosin. Six to 8 sections at a distance of 84 μm were analyzed for eachspecimen to insure representative results. Duplicate slides weredeveloped with monoclonal antibodies mAb-4E6, specific for oxidized LDL,PG-M1, specific for human macrophages, or 1A4, specific for human smoothmuscle α-actin (both from Dako S A, Glostrup, Denmark). Specificity ofbinding of mAb-4E6 was confirmed by its inhibition with OxLDL but notwith native LDL.

3. Results

Coronary artery segments of 7 individuals with pretransplant dilatedcardiomyopathy did not contain atherosclerotic lesions and themonoclonal antibody did not detect OxLDL and/or aldehyde-modified LDL inthese segments. Coronary artery segments of 7 patients withpretransplant ischemic heart disease all contained atheroscleroticlesions which contained OxLDL and/or aldehyde-modified LDL (FIG. 2).This information is sufficient to state that the antibody detects OxLDLin atherosclerotic lesions in a highly specific manner.

OxLDL was associated with macrophage foam cells (preferentially inlesions with <50% stenosis), with smooth muscle foam cells and with thenecrotic lipid core (preferentially in lesions with >50% stenosis).Macrophages and smooth muscle cells were identified by immunostainingwith specific monoclonal antibodies (5). These data supported thehypothesis that oxidation of LDL may be associated with the developmentof ischemic coronary artery disease. The monoclonal antibody mAb-4E6 ofthe present invention that detected the immunoreactive material in thetissue sections was then further used in ELISA (cf. Example 4).

4. Legend to FIG. 2

Light micrographs (a, c, e; ×40) and phase contrast micrographs (b, d,f; ×400) of representative left anterior descending coronary arteryspecimens of a patient with dilated cardiomyopathy (male; 40 years ofage) (a, b) and of a patient with ischemic heart disease (male; 57 yearsof age) (c-f). Tissue sections were immunostained with the monoclonalantibody mAb-4E6. Oxidized LDL was undetectable in the neointima of thefirst patient (a, b), but demonstrable in plaques of the second patient.The oxidized LDL was associated with macrophage foam cells thatinfiltrated at the shoulder areas of fibrous plaques (c, d) and withsmooth muscle foam cells in fibrous caps (e, f).

Example 4

Competitive ELISA

1. Introduction

According to the invention an ELISA was established for the quantitationof OxLDL and aldehyde-modified LDL in plasma. It was based on theinhibition of the binding of mAb-4E6 to the wells of microtiter platescoated with copper-oxidized LDL. This antibody was obtained as describedin Example 1.

2. Material and Methods

Standard OxLDL and aldehyde-modified LDL and plasma samples were dilutedin PBS containing 1 mM EDTA, 20 μM vitamin E, 10 μM butylatedhydroxytoluene, 20 μM dipyridamole and 15 mM theophylline to prevent invitro LDL oxidation and platelet activation. Equal volumes of dilutedpurified mAb-4E6 solution (final concentration 7.5 ng/ml) and of dilutedstandard solution (copper-oxidized LDL added as competing ligand at afinal concentration ranging from 50 to 500 ng/ml) were mixed andincubated for 30 min at room temperature. Then 200 μl aliquots of themixtures were added to wells coated with MDA-modified LDL or OxLDL.

Samples were incubated for 2 h at room temperature. After washing, thewells were incubated for 1 h with horse-radish peroxidase conjugatedrabbit IgG raised against mouse immunoglobulins and washed again. Theperoxidase reaction was performed as described earlier (5) and theabsorbance (A) was read at 492 nm.

Controls without competing ligand and blanks without antibody wereincluded routinely. The percent inhibition of binding of mAb-4E6 to theimmobilized ligand was calculated as:$\frac{{A^{492\quad{nm}}{control}} - {A^{492\quad{nm}}{sample}}}{{A^{492\quad{nm}}{control}} - {A^{492\quad{nm}}{blank}}}$and standard curves were obtained by plotting the percentage ofinhibition vs the concentration of competing ligand.

The lower limit of detection was 0.020 mg/dl in undiluted human plasma.Intra- and interassay coefficients of variation were 10 and 12%,respectively. Standard OxLDL and aldehyde-modified LDL and plasmasamples were diluted in PBS containing antioxidants and antiplateletagents as described above.

3. Results

The specificity of mAb-4E6 for OxLDL and aldehyde-modified LDL isillustrated in FIG. 3. 50% inhibition of binding of mAb-4E6 toimmobilized OxLDL and aldehyde-modified LDL was obtained with 0.025mg/dl copper-oxidized LDL and 25 mg/dl native LDL, respectively. The C₅₀value, i.e., the concentration that is required to obtain 50% inhibitionof antibody binding, increased from 2.5 mg/dl for MDA-modified LDL with60 substituted lysine residues per apo B-100 molecule to 0.025 mg/dl forMDA-modified LDL with 240 substituted lysine residues per apo B-100molecule (FIG. 3). Copper-oxidation resulted in fragmentation of the apoB-100 moiety but did not abolish the binding of mAb-4E6 (FIG. 3).50-fold higher molar concentrations of MDA-modified albumin wererequired to obtain 50% inhibition (not shown), whereas up to 1,000-foldhigher molar concentrations of MDA-modified lysine did not affectmAb-4E6 binding. OxLDL and aldehyde-modified LDL isolated from patientplasma had the same reactivity as MDA-modified LDL with 120 substitutedlysines and as copper-oxidized LDL with 210 substituted lysines. Intra-and interassay coefficients of variation were 10 and 12%, respectively.When copper-oxidized LDL were added to human plasma at a finalconcentration of 0.25 and 2 mg/dl, respectively, recoveries were 95 and105%, respectively.

4. Legend to FIG. 3

Interaction of mAb-4E6 with competing ligands in solution.Copper-oxidized LDL (1 μg/ml) was the plated antigen. mAb-4E6 was addedin the absence and in the presence of competing ligands: copper-oxidizedLDL (∇), MDA-modified LDL with 240 (▪), 120 (⋄), 90 (∘) and 60 (●)blocked or substituted or modified lysines per apo B-100, respectively,native LDL (▴), and OxLDL and aldehyde-modified LDL (♦) isolated fromthe plasma of severe chronic renal failure patients. Results areexpressed as B/B₀ where B₀ is the amount of mAb-4E6 bound in the absenceand B that amount bound in the presence of competing ligand.

Example 5

Sandwich ELISA

1. Introduction

According to the invention a sandwich-type ELISA was established for thequantitation of OxLDL and aldehyde-modified LDL in plasma. It was basedon the binding of immunoreactive material to the wells of microtiterplates coated with the monoclonal antibody mAb-4E6 and the detection ofbound immunoreactive material with the use of the monoclonal antibodymAb-8A2 labeled with peroxidase. This version of the ELISA is moresuited for use in the clinical laboratory because it overcomes the needto prepare standard solutions of in vitro oxidized and/oraldehyde-modified LDL which can only be kept at −4° C. for a limitedperiod of time, typically 2 weeks. MDA-modified LDL may be added toreference plasma and those standard preparations may be stored at −80°C. for up to 1 year (see above).

2. Material and Methods

Standard preparations and plasma samples were diluted in PBS containingantioxidants and antiplatelet agents as described above, 180 μl aliquotsof 80-fold diluted plasma and of standard solutions containing between10 and 0.01 nM of malondialdehyde-modified LDL were applied to the wellsof microtiter plates coated with mAb-4E6 (200 μl of a 4 μg/ml IgGsolution) and incubated for 2 h at room temperature. After washing, thewells were incubated for 1 h with horseradish peroxidase conjugatedmAb-8A2, IgG (final IgG concentration 65 ng/ml) and washed again. Theperoxidase reaction was performed as described above. The absorbancemeasured at 492 nm correlates with the log-value of thealdehyde-modified LDL concentration in the range between 1.5 nM and 0.3nM.

3. Legend to FIG. 4

Standard curves for the sandwich ELISA mAb-4E6 was the plated antibody.MDA-modified LDL was the ligand. Bound MDA-modified LDL was detectedwith mAb-8A2 conjugated to horse radish peroxidase. MDA-modified LDL wasadded to 8 different plasma samples to a final concentration of 100 nMand further diluted in buffer to final concentrations ranging from 2 to0.2 nM.

Example 6

Use of the ELISA in Diagnosis of Posttransplant Coronary Artery Disease

1. Introduction

The ELISA of the present invention was used to study the associationbetween plasma levels of OxLDL and aldehyde-modified LDL andposttransplant coronary artery disease.

2. Material and Methods

2.1. Patients

The posttransplant study group contained 47 patients transplanted fordilated cardiomyopathy and 60 patients treated for ischemic heartdisease. The clinical characteristics of these patients are summarizedin Table 1. At the time of blood sampling, between 12 and 84 monthsafter surgery, all patients were in a stable cardiac condition withoutevidence of acute rejection. From 14 patients (7 dilated cardiomyopathyand 7 ischemic heart disease patients) coronary arteries of cardiacexplants were isolated and studied by immunohistochemistry (asdemonstrated in Example 3). Adequate information about smoking habitswas available for 92 of the 107 patients (16 smokers and 76non-smokers). There was no adequate information about smoking habits ofdonors. Blood samples of 53 non-smoking controls (25 males/28 females;age: 52±1.3 years) without a history of atherosclerotic cardiovasculardisease were obtained. The controls were matched for age, gender andlevels of LDL cholesterol. They were selected from the laboratory andclinical staff.

2.2. Coronary Angiography

Routine annual coronary angiograms were available for all posttransplantpatients at the time of blood sampling. Coronary artery disease wasindependently assessed by two angiographers who where unaware of theOxLDL and aldehyde-modified LDL levels and was visually graded asfollows:

-   -   grade 0: normal coronary arteries    -   grade I: minor abnormalities with <50% stenosis of primary or        secondary branches and normal left ventricular function    -   grade II: ≧50% stenosis of primary or secondary branches, or        distal involvement with impaired left ventricular function.        It is well known that angiography systematically underestimates        the extent of coronary intimal thickening in cardiac transplant        recipients. This study therefore does not attempt to accurately        quantify the coronary artery disease in our patients. Rather the        subdivision in groups defined above relies on angiographic data        that are easily distinguishable and that have been shown to        correlate with histopathologic findings. Out of 107 patients, 46        patients had a normal coronary angiogram 3 years before and        development of angiographic, coronary artery disease within a 3        year follow-up period was assessed in all these patients. The        reference normal coronary angiogram was the first post-operative        angiogram in 18 patients, the second in 14 patients and the        third in 14 patients.

The study was approved by the Institutional Review Board and the studysubjects provided informed consent.

2.3. Blood Sampling

Venous blood samples from patients and controls were collected on 0.1vol of 0.1 M citrate, containing 1 mM EDTA, 20 μM vitamin E, 10 μMbutylated hydroxytoluene, 20 μM dipyridamole and 15 mM theophylline toprevent in vitro LDL oxidation and platelet activation. Blood sampleswere centrifuged at 3,000 g for 15 min at room temperature within 1 h ofcollection and stored at −20° C. until the assays were performed.

2.4. Lipoproteins: Isolation and Modification

LDL were isolated from pooled sera of fasting normolipidemic donors bydensity gradient ultra-centrifugation (document 6). Standardpreparations of MDA-modified and copper-oxidized LDL were prepared asdescribed elsewhere (7, 8) and were used as assay controls. Apo B-100molecules of in vitro MDA-modified LDL (7) and of copper-oxidized LDL(8) contained on average 244, and 210 substituted lysines (out of atotal of 356), respectively (5, 9). Whereas the extent oflysine-substitution in in vitro MDA-modified LDL and copper-oxidized LDLis very similar, the lipid moiety of the former is not oxidized.Specificity of the monoclonal antibody mAb-4E6 for both MDA-modified LDLand copper-oxidized LDL suggests that it depends on the extent ofprotein (lysine) modification only. All lipoprotein concentrations weretherefore expressed in terms of protein. OxLDL and aldehyde-modified LDLisolated from the plasma of patients were characterized as describedpreviously (5, 9).

2.5. Assays

Cholesterol and triglycerides were measured by enzymatic methods(Boehringer Mannheim, Meylon, France). Typing of majorhistocompatibility complex class I (HLA-B) and class II (HLA-DR) antigenwas performed by the microlymphocytotoxicity technique.

The ELISA of the invention was used to detect OxLDL andaldehyde-modified LDL.

2.6. Statistical Analysis

Controls and patients were compared by ANOVA test followed bynonparameteric Mann-Whitney or Dunnett's multiple comparison test onlogarithmically transformed values, in the Instat V2.05a statisticalprogram (Graph Pad Software, San Diego, Calif.). Non-quantitativeparameters were compared by Chi-square analysis. OxLDL andaldehyde-modified LDL levels measured in 3 aliquots of the same plasmasamples were compared in Friedman nonparametric repeated measures test.Logistic regression analysis, using the SAS software (SAS InstituteInc., USA), was performed to evaluate the correlation betweenangiographically assessed coronary artery stenosis (as dependentvariable) and plasma levels of OxLDL and aldehyde-modified LDL, age andsex of recipients, age and sex of donors, pretransplant history ofischemic heart disease or dilated cardiomyopathy, duration of ischemia,length of follow up, number of rejections, number of HLA-mismatches,cytomegalovirus infection, hypertension (antihypertensive treatment),diabetes, treatment with lipid lowering drugs (statins or fibrates) andserum levels of LDL cholesterol, HDL cholesterol and triglycerides asindependent variables. p-values of less than 0.05 were considered toindicate statistical significance. Logistic regression analysis was alsoperformed to evaluate the correlation between plasma levels of OxLDL andaldehyde-modified LDL and development of coronary artery stenosis duringa 3-year follow-up period.

3. Results

The correlation between OxLDL and aldehyde-modified LDL and coronaryartery stenosis was evaluated in 47 patients transplanted for dilatedcardiomyopathy and in 60 patients treated for ischemic heart disease.Analysis of clinical data for the two groups of heart transplantpatients (Table 1) revealed no significant differences in age and genderof the recipients, age and gender of donors, duration of ischemia of thedonor heart, number of rejection episodes, number of HLA-mismatches,frequency of Cytomegalovirus infections, hypertension or diabetes, andgrade of coronary artery stenosis. Patients transplanted for ischemicheart disease were followed longer and received more frequently lipidlowering drugs (Table 1).

Analysis of the laboratory data (Table 2) revealed no significantdifferences in serum levels of triglycerides, HDL cholesterol and LDLcholesterol between groups of patients or between patients and controls.However, significant differences in levels of OxLDL andaldehyde-modified LDL were observed. Mean plasma levels of OxLDL andaldehyde-modified LDL were 1.3±0.14 mg/dl in dilated cardiomyopathypatients (p<0.001 vs controls) and 1.7±0.13 mg/dl in ischemic heartdisease patients (p<0.001 vs controls and <0.01 vs dilatedcardiomyopathy patients) (Table 2). Plasma levels of OxLDL andaldehyde-modified LDL in control subjects matched for age, gender andserum levels of triglycerides, HDL cholesterol and LDL cholesterol were0.60±0.034 mg/dl (n=53; p<0.001 vs both transplanted dilatedcardiomyopathy and ischemic heart disease patients).

Levels of OxLDL and aldehyde-modified LDL were not different in samplesthat were stored for 24 h to 4 months after collection, and up to fourthawing and freezing cycles did not cause an increase of OxLDL andaldehyde-modified LDL levels. These findings indicated that the additionof EDTA, antioxidants and anti-platelet agents adequately prevented thein vitro oxidation of LDL. In a subset of 87 consecutive plasma sampleslevels of OxLDL and/or aldehyde-modified LDL were measured in 3 separatealiquots on 3 different days. The levels were 1.30±0.074 mg/dl,1.48±0.101 mg/dl and 1.46±0.090 mg/dl, respectively. Friedmannonparametric repeated measures test revealed no significantdifferences.

Mean OxLDL and aldehyde-modified LDL levels were 1.2±0.053 mg/dl (n=79)in posttransplant samples of patients with angiographically normalcoronary arteries (grade 0), 2.1±0.30 mg/dl in patients with grade Icoronary artery stenosis (n=18; p<0.001 vs grade 0) and 3.2±0.45 mg/dlin patients with grade II coronary artery stenosis (n=10; p<0.001 vsgrade 0 and p<0.05 vs grade I) (FIG. 5). Serum levels of LDLcholesterol, triglycerides and HDL cholesterol were very similar inpatients with higher grade of coronary artery stenosis. Levels of OxLDLand aldehyde-modified LDL in plasma samples of patients transplanted fordilated cardiomyopathy or ischemic heart disease, with the same grade ofcoronary artery stenosis, were similar: 1.1±0.072 and 1.4±0.079 mg/dlfor grade 0 patients and 2.6±0.60 and 2.4±0.29 mg/dl for patients withhigher grade of coronary artery stenosis. The number of patients withelevated levels of OxLDL and aldehyde-modified LDL (>1 mg/dl, i.e. meanlevels of controls+2 SD) were 43 (out of 60) in the subpopulation ofpatients with pretransplant ischemic heart disease and 21 (out of 47) inthe subpopulation of patients with pretransplant dilated cardiomyopathy.Forty-two out of 79 patients with angiographically normal coronaryarteries had elevated levels of OxLDL and aldehyde-modified LDL.Elevated levels were detected in 12 (out of 18) patients with grade Iand in all patients with grade II stenosis (p=0.0046 for trend).

To allow further characterization of the immunoreactive materialdetected in the ELISA, LDL fractions were isolated from the plasma ofall of 10 patients with grade II coronary artery stenosis (18). Thesefractions retained 85±10% (mean±SD) of the immunoreactive material,whereas no immunoreactive material migrated in the serum albuminposition. OxLDL and aldehyde-modified LDL were isolated from isolatedLDL fractions by ion-exchange chromatography on a mono Q-Sepharosecolumn with a recovery of 75%. The number of substituted lysines per apoB-100 molecule was 130±10 for OxLDL and aldehyde-modified LDL comparedto 5±1 (p<0.001) for native LDL. The respective cholesterol/proteinratios were 3.3±0.54 and 1.8±0.36 (p<0.001). The levels of arachidonateand linoleate in OxLDL and aldehyde-modified LDL isolated from theplasma of these patients were 75 and 80% lower than these in native LDLisolated from the same plasma samples. The inhibition curves obtainedwith OxLDL and aldehyde-modified LDL isolated from the plasma of hearttransplant patients were superimposable with these obtained with invitro oxidized LDL with the same extent of protein modification (120substituted lysines per apo B-100 molecule) (FIG. 3).

The protein/antigen ratio and the relative reactivity in the ELISA ofOxLDL and aldehyde-modified LDL isolated from the plasma of thesepatients were similar to these of copper-oxidized or MDA-modifiedstandard LDL preparations.

Logistic regression analysis (Table 3) identified 3 parameters that weresignificantly and independently correlated with posttransplant coronaryartery stenosis including levels of OxLDL and aldehyde-modified LDL,length of follow up and donor age.

In contrast, pretransplant history of dilated cardiomyopathy or ofischemic heart disease, age and gender of recipients, gender of donors,duration of ischemia of the donor heart, extent of HLA-mismatch, numberof rejections, hypertension, diabetes, and serum levels of LDLcholesterol, HDL cholesterol and triglycerides in recipients did notsignificantly contribute to the individual variations in extent ofcoronary artery stenosis (Table 3).

Serum levels of LDL cholesterol, HDL cholesterol and triglycerides inpatients were similar to these in controls (Table 2), so that highergrade of coronary artery stenosis was unlikely to depend on thesevariables in this study group. Fifty-six of the 107 transplant patientsreceived lipid lowering drugs (46 with statins and 10 with fibrates)(Table 1), but the treatment with these drugs was not correlated withthe incidence of angiographic graft vasculopathy (Table 3). Seventy-five(out of 107) patients were treated with calcium channel blockers. Theplasma levels of OxLDL and aldehyde-modified LDL in these patients(1.53±0.11 mg/dl) were very similar to these in non-treated patients(1.74±mg/dl) and treatment with these drugs was not correlated with theextent of coronary artery stenosis.

Development of coronary artery disease was observed in 12 out of 46heart transplantation patients during a 3-year follow-up period. Therewere no differences in age and gender of recipients, age and gender ofdonors, duration of ischemia, extent of HLA mismatch, frequency ofcytomegalovirus infections, hypertension and diabetes (Table 4) nor inserum levels of triglycerides, HDL cholesterol and LDL cholesterol(Table 5) between patients without and with development of coronaryartery disease. However, levels of OxLDL and aldehyde-modified LDL weresignificantly elevated in patients with development of coronary arterydisease (Table 5).

Logistic regression analysis revealed that plasma levels of OxLDL andaldehyde-modified LDL (Chi-square value=7.1; p=0.0076) and age of donor(Chi-square value=4.4; p=0.035) predicted the development of coronaryartery disease in these patients. Three of these patients developedcoronary artery disease in the first year, 3 in the second and 6 in thethird year. The plasma levels of OxLDL and aldehyde-modified LDL were3.9±0.6 mg/dl, 2.0±0.37 mg/dl and 1.2±0.33 mg/dl, respectively. Althoughstatistical analysis showed no correlation with gender, hypertension andCytomegalovirus infection, 8 out of 12 of these patients were male,hypertensive and had Cytomegalovirus infection.

4. Discussion

This demonstrates:

1) that cardiac explants of patients with ischemic heart disease, butnot with dilated cardiomyopathy, contain oxidized LDL in macrophages andin smooth muscle cells in atheromatous plaques;

2) that posttransplant coronary artery disease is associated withincreased plasma levels of OxLDL and aldehyde-modified LDL both inpatients transplanted for dilated cardiomyopathy or for ischemic heartdisease, and

3) that increased plasma levels of OxLDL and aldehyde-modified LDLcorrelate with the development of coronary artery stenosis.

OxLDL and aldehyde-modified LDL levels in plasma samples of hearttransplant patients without angiographically detectable coronary arterylesions were 2-fold higher than in plasma samples of control subjectswithout a history of atherosclerotic cardiovascular disease, who werematched for age, gender, and plasma levels of LDL cholesterol, HDLcholesterol and triglycerides. A further 2.7-fold increase was observedin posttransplant plasma samples of patients with pronounced coronaryartery stenosis. These data suggest that elevated plasma levels of OxLDLand aldehyde-modified LDL may be an indicator of posttransplant coronaryartery stenosis. Increased plasma levels of OxLDL and aldehyde-modifiedLDL correlated with the extent of coronary artery stenosis and also withits progression, suggesting that OxLDL and aldehyde-modified LDL mayplay a pathogenic role in the accelerated progression of coronary arterydisease in heart transplant patients.

It has been suggested that posttransplant atherosclerosis results from a“response to injury” of the endothelium (10). The extent of ischemicinjury in endomyocardial biopsies was indeed found to be a strongpredictor of the development of accelerated atherosclerosis (11-13).Endothelial injury may be induced by cellular delayed-typehypersensitivity immune responses elicited by class IIhistocompatibility (HLA) antigens on coronary artery endothelium (14),by cytomegalovirus infection (15, 16), by cyclosporin (17) and by OxLDLand aldehyde-modified LDL (18) that may act synergistically withcyclosporin (19). In the present study, the extent ofhistoincompatibility between pairs of donors and recipients, the numberof episodes of rejection or Cytomegalovirus infection did not correlatewith the grade of coronary artery stenosis, whereas OxLDL andaldehyde-modified LDL were significantly and independently correlatedwith posttransplant coronary artery disease. The observed associationbetween the age of the donor and the occurrence of coronary arterydisease is in agreement with previous findings that coronaryatherosclerosis in the donor heart predisposes to acceleratedposttransplant coronary artery stenosis (20).

OxLDL and aldehyde-modified LDL were demonstrated in coronary arteriesin cardiac explants of ischemic heart disease patients suggesting thatOxLDL and aldehyde-modified LDL that accumulate in the arterial wall maycontribute to the progression of coronary artery stenosis. Thecholesterol/protein ratio in OxLDL and aldehyde-modified LDL was verysimilar to that in LDL extracted from atherosclerotic lesions asdescribed previously (21,22). A possible explanation is that at leastpart of the OxLDL and aldehyde-modified LDL is released from thearterial wall. Previously, we have demonstrated that plaque rupture inacute myocardial infarction patients is associated with the release ofoxidatively modified LDL (5).

In vitro data suggest that OxLDL and aldehyde-modified LDL may be linkedto atherogenesis by a sequence of events (reviewed in 2,23). Endothelialcells exposed to OxLDL and aldehyde-modified LDL secrete adhesionmolecules, chemoattractant proteins and colony-stimulating factors thatenhance the infiltration, proliferation and accumulation ofmonocytes/macrophages in the arterial wall. Uptake of OxLDL andaldehyde-modified LDL by infiltrated macrophages may result in thegeneration of foam cells that produce oxygen radicals and thus furthercontribute to the oxidation of LDL. It has been demonstrated that OxLDLand aldehyde-modified LDL inhibit the migration of aortic endothelialcells in vitro, suggesting that OxLDL and aldehyde-modified LDL maylimit the healing response of the endothelium after injury, and thatbasic fibroblast growth factor reverses the atherosclerosis associatedimpairment of human coronary angiogenesis-like responses in vitro(24,25). OxLDL and aldehyde-modified LDL may also contribute to rapidlyprogressing coronary atherosclerosis by inducing platelet adhesion, bydecreasing the anticoagulant and fibrinolytic capacities of activatedendothelium and by impairing vasodilation and inducing shear stress(2,23).

Increased intracellular levels of ferritin (26) or of alpha-tocopherolanalogs (27) decreased the extent of endothelial injury elicited byOxLDL and aldehyde-modified LDL in vitro, whereas antioxidants protectagainst progression of atherosclerosis in experimental animals (reviewedin document 28).

In summary, the present example demonstrates that posttransplantatherosclerosis correlates with plasma levels of OxLDL andaldehyde-modified LDL.

5. Legend to the FIG. 5

Plasma levels of OxLDL and aldehyde-modified LDL and angiographicallyassessed grade of coronary artery stenosis. Grade 0: normal coronaryarteries; grade I: minor abnormalities with <50% stenosis of primary orsecondary branches and normal left ventricular function; and grade II:≧50% stenosis of primary or secondary branches, or distal occlusionswith impaired left ventricular function.

Example 7

Use of the ELISA in Renal Failure Patients

1. Material and Methods

1.1. Subjects

The patient population consisted of 20 mild chronic renal failure (MCRF)and 77 severe chronic renal failure patients: 21 on conservativetreatment including dietary and antihypertensive treatment (SCRF), and56 on a four-hour, three times a week hemodialysis schedule (HEMO) for66 months (95% CI, 50-82 months). All hemodialysis patients were givenan oral polyvitamin preparation (Ol-Amine, La Meuse, Belgium) afterhemodialysis, which contained only minute amounts of antioxidantcompounds (i.e. 5 mg of vitamin E and 100 mg of vitamin C). Controls andnon-dialyzed patients did not receive routine prescriptions of vitaminsupplements. The high frequency of atherosclerotic disease in thesepatients (Table 6) is in agreement with previously published data (29,30). The diagnosis of atherosclerotic heart disease, cerebrovasculardisease and peripheral vascular disease was made after reviewing thepatient files for a history of myocardial infarction, unstable angina orantianginal treatment, cerebrovascular accidents, transient ischemicattack or events related to peripheral vascular disease such as ischemiculcera, amputation or bypass surgery. Angiograms were available for onlya few patients. No patients had evidence of unstable atheroscleroticdisease at the time of blood sampling nor in the following days. A groupof 27 healthy volunteers (Table 6) without a history of renal disease oratherosclerotic vascular disease served as controls. Patients receivinglipid lowering drugs were excluded. The study was approved by theInstitutional Review Board and the study subjects provided informedconsent.

1.2. Blood Samples

Venous blood samples from patients and controls were collected on 0.1vol of 0.1 M citrate, containing 1 mM EDTA, 20 μM vitamin E, 10 μMbutylated hydroxytoluene, 20 μM dipyridamole and 15 mM theophylline toprevent in vitro LDL oxidation and in vitro platelet activation,respectively. Blood samples were centrifuged at 3,000 g for 15 min atroom temperature within 1 h of collection and stored at −20° C. untilthe assays were performed.

1.3. Assays

Titers of autoantibodies against OxLDL and aldehyde-modified LDL andnative LDL were measured according to Salonen et al. (3) as described indetail elsewhere (5). vWF antigen levels were measured in asandwich-type ELISA based on a polyclonal rabbit anti-human vWFantiserum (Dako, Glostrup, Denmark), horseradish peroxidase-conjugatedrabbit anti-human vWF IgG (Dako) and o-phenylenediamine. Plasma levelsof total cholesterol, HDL cholesterol and triglycerides were determinedusing standard enzymatic assays (Boehringer Mannheim, Meylon, France).The LDL cholesterol levels were calculated using the Friedewald formula.For the patients not in hemodialysis, creatinine clearance rates werecalculated from plasma creatinine levels using the Cockcroft and Gaultformula (31).

1.4. Statistical Analysis

Controls and patients were compared by ANOVA test followed by Dunnett'smultiple comparison test, in the Instat V2.05a statistical program(Graph Pad Software, San Diego, Calif.). Correlation coefficients werecalculated according to Spearman. Multiple regression analysis, usingthe SAS software (SAS Institute Inc., USA), was performed to study therelationship between OxLDL and aldehyde-modified LDL as dependentvariable, and age, sex, hypertension (antihypertensive treatment),levels of triglycerides, HDL cholesterol, LDL cholesterol and creatinineclearance rates (marker of extent of renal failure) and levels of vWF(marker of endothelial injury) as independent variables.

2. Results

Mean plasma levels of OxLDL and aldehyde-modified LDL in controls were0.59 mg/dl (95% CI, 0.52-0.66 mg/dl; n=27), and were 2.7-fold higher inMCRF patients (p<0.01 as determined by Dunnett's multiple comparisontest), 3.1-fold higher in SCRF patients (p<0.001), and 5.4-fold higherin HEMO patients (p<0.001) (Table 7). OxLDL and aldehyde-modified LDLlevels were inversely correlated with creatinine clearance rates(r=−0.65; p<0.001; n=73). HEMO patients were not included in thisanalysis because their plasma creatinine clearance cannot be determinedadequately.

In a series of 14 hemodialyzed patients, levels of OxLDL andaldehyde-modified LDL were found to be very similar in fresh and infresh frozen plasma samples. Three freezing and thawing cycles did notcause an increase of OxLDL and aldehyde-modified LDL, indicating thataddition of antioxidants and antiplatelet agents prevented in vitrooxidation.

Plasma samples were obtained from 14 hemodialyzed patients on 3consecutive days before the start of the dialysis procedure. The levelsof OxLDL and aldehyde-modified LDL in these samples were similar:3.4±0.25 mg/dl, 3.2±0.21 mg/dl and 3.5±0.28 mg/dl, respectively.Furthermore, plasma samples were obtained during (after 2 h) and at theend (after 4 h) of hemodialysis. Plasma levels of OxLDL andaldehyde-modified LDL were 4.0±0.60 mg/dl and 4.7±0.70 mg/dl (p=NS vsbefore) as compared to 3.4±0.25 mg/dl before the start of the dialysisprocedure. Thus the hemodialysis procedure did not induce a significantincrease in the OxLDL and aldehyde-modified LDL levels.

Adequate information about smoking habits was only available forcontrols (27 non-smokers) and for HEMO patients (12 smokers and 45non-smokers). Levels of OxLDL and aldehyde-modified LDL were somewhathigher in smoking HEMO patients (3.6 mg/dl; 95% CI, 2.1-5.6 mg/dl) thanin non-smoking HEMO patients (3.0 mg/dl; 95% CI, 2.5-3.6 mg/dl; p=NS).The plasma levels of OxLDL and aldehyde-modified LDL in hemodialyzedpatients with a history of unstable atherosclerotic cardiovasculardisease were 3.5±0.40 mg/dl (n=30) as compared to 2.8±0.60 mg/dl (n=26,p=NS) in hemodialyzed patients without a history of unstableatherosclerotic cardiovascular disease.

LDL fractions were isolated from the plasma of 10 controls, of 10 MCRFpatients, of 10 SCRF-patients and of 10 HEMO patients by gel filtrationon a Superose 6HR 10/30 column, as described previously (5). 75±6%(mean±SD), 80±4%, 83±6% and 79±5% of the immunoreactive material wasrecovered in the LDL fractions. No immunoreactive material migrated inthe serum albumin position. The inhibition curves obtained with therespective LDL fractions were parallel to those obtained with in vitrocopper-oxidized or MDA-modified standard LDL preparations. OxLDL andaldehyde-modified LDL were isolated from isolated LDL fractions of 10SCRF patients by ion-exchange chromatography on a mono Q-Sepharosecolumn with a recovery of 75%. Their physicochemical properties aresummarized in Table 8. The levels of arachidonate of OxLDL andaldehyde-modified LDL isolated from these patients were reduced with75%, whereas its linoleate levels were reduced with 80%. Thirty-seven %of the lysine residues of OxLDL were substituted with aldehydes. Theinhibition curves obtained with OxLDL and aldehyde-modified LDL isolatedfrom the plasma of chronic renal failure patients were parallel to theseobtained with OxLDL and aldehyde-modified LDL that was obtained by invitro oxidation of LDL that had been isolated from the plasma of controlsubjects (FIG. 3). The protein/antigen ratio and the relative reactivityin the ELISA of OxLDL and aldehyde-modified LDL isolated from the plasmaof these patients were similar to these of copper-oxidized orMDA-modified standard LDL preparations (Table 8).

Titers of autoantibodies against OxLDL and aldehyde-modified LDL were4.2 (95% CI, 4.0-4.4) in controls, were similar in MCRF and SCRFpatients, but significantly increased in HEMO patients (p<0.001) (Table7). Autoantibody titers correlated with levels of OxLDL andaldehyde-modified LDL in SCRF patients (r=0.44; p=0.047) and in HEMOpatients (r=0.37; p=0.0055) (FIG. 6). No circulating autoantibodiesagainst native LDL could be detected.

Levels of vWF were 100 percent in controls (95% CI, 90-110 percent), andwere 1.5-fold higher in MCRF patients (p=NS vs controls), 1.6-foldhigher in SCRF patients (p<0.01) and 2.1-fold higher (p<0.001) in HEMOpatients (Table 7). Levels of vWF were not significantly higher insmoking HEMO patients (250 percent; 95%, 150-340 percent; n=12) than innon-smoking HEMO patients (220 percent; 95% CI, 190-260 percent; n=45).Levels of vWF correlated with levels of OxLDL and aldehyde-modified LDLin MCRF patients (r=0.59; p<0.0057), in SCRF patients (r=0.69; p=0.0006)and in HEMO patients (r=0.62; p<0.0001) (FIG. 7). In contrast, levels ofvWF did not correlate with LDL cholesterol levels or with body weight.

Multiple regression analysis revealed that the extent of renal failure(F=14; p=0.0004) and the extent of endothelial injury (F=26; p=0.0001),but not age, sex, hypertension, triglyceride levels, HDL cholesterol orLDL cholesterol levels, accounted for a significant fraction of thevariations in OxLDL and aldehyde-modified LDL levels (Table 9). Evenwhen only subjects without evidence of ischemic atherosclerotic disease(n=53) were included in the model (R²-value=0.68) only the extent ofrenal failure (F=21; p=0.0001) and the extent of endothelial injury(F=14; p=0.0006) contributed significantly to the variations in OxLDLand aldehyde-modified LDL levels. No other variables contributedsignificantly to these variations after exclusion of subjects withoutevidence of ischemic atherosclerotic disease. When only subjects withevidence of ischemic atherosclerotic disease (n=15) were included onlythe extent of endothelial injury (F=6.2; p=0.047; R²-value=0.65)contributed to the variations in OxLDL and aldehyde-modified LDL levels.Exclusion of diabetic patients did not significantly change the dataeither. After exclusion of the extent of renal failure as an independentvariable, multiple regression analysis revealed that hemodialysis(F=5.6; p=0.021; n=77), LDL cholesterol levels (F=7.1; p=0.0095) andendothelial injury (F=35; p=0.0001) accounted for a significant fractionof the variation in OxLDL and aldehyde-modified LDL levels in severechronic renal failure patients (Table 10).

3. Discussion

In vitro work and experimental animal data suggest that oxidized LDL(OxLDL and aldehyde-modified LDL) may contribute to the progression ofatherosclerosis (reviewed in document 2), and OxLDL andaldehyde-modified LDL have been demonstrated in human atheroscleroticplaques (5). The immuno-assay of this invention identifies OxLDL andaldehyde-modified LDL (MDA-modified LDL) with ≧60 substituted lysinesper apo B-100 molecule, which represents the threshold of substitutionrequired for scavenger receptor mediated uptake (1). Increased levels ofOxLDL and aldehyde-modified LDL have been measured by ELISA in theplasma of chronic renal failure patients.

Overall, 80 percent of the immunoreactive material isolated from theplasma of patients was recovered in the LDL fractions that wereseparated by gel filtration. No immunoreactive material migrated in thealbumin position. Inhibition curves obtained with the isolated OxLDL andaldehyde-modified LDL were parallel to these of in vitro copper-oxidizedor MDA-modified LDL standard preparations and the protein/antigen ratioand the C₅₀ value of the isolated OxLDL and aldehyde-modified LDL wereidentical to these of standard OxLDL and aldehyde-modified LDLpreparations. These data suggested that increased immunoreactivity ofOxLDL and aldehyde-modified LDL fractions in plasma of these patientswith the antibodies of this invention depended indeed on the higherextent of protein modification and not on changes in lipid compositionas was previously observed with other antibodies (32). The increasedelectrophoretic mobility, the increased lysine modification, theincreased cholesterol/protein ratio, the decreased arachidonic acid andlinoleate levels were very similar to these of modified LDL extractedfrom atherosclerotic lesions (21, 22). OxLDL and aldehyde-modified LDLinduced foam cell generation, suggesting that OxLDL andaldehyde-modified LDL were not “minimally modified” LDL.

Multiple regression analysis revealed that chronic renal failure andendothelial injury contributed significantly to the variation in OxLDLand aldehyde-modified LDL levels even when patients with evidence ofischemic atherosclerotic disease were excluded. Indeed, 79.6% and 82.4%of the variations in OxLDL and aldehyde-modified LDL levels could beexplained in these models. No patients had evidence of unstableatherosclerotic disease at the time of blood sampling nor in thefollowing days and exclusion of patients with a history of ischemicatherosclerotic disease did not affect the contribution of the extent ofrenal failure and of endothelial injury to the variations in OxLDL andaldehyde-modified LDL.

LDL cholesterol levels in controls and patients were very similar andLDL cholesterol levels did not contribute to the variations in OxLDL andaldehyde-modified LDL levels. Sutherland et al. (33) demonstrated thatthe lag time of conjugated diene formation, which is a measure for thesensitivity of LDL to in vitro oxidation, was similar in patients withchronic renal failure and in matched controls. The maximum rate and theextent of LDL oxidation were even lower in patients with renal diseasethan in controls, due to lower levels of linoleic acid and higher levelsof oleic acid. Furthermore, Schulz et al. (34) demonstrated that despitethe fact that hemodialysis causes leukocyte activation, the in vitro LDLoxidation lag time was similar in renal patients and in healthycontrols. It was concluded that the antioxidative defense oflipoproteins was preserved in renal failure and during dialysis.

In experimental models, antioxidants such as probucol and vitamin E werefound to protect against glomeral injury (35, 36) and to slowatherogenic processes (28). Renal vasoconstriction caused by cholesterolfeeding was corrected by probucol or by a thromboxane antagonist (35).Galle et al. (38) demonstrated that the inhibition ofendothelium-dependent dilation induced by oxidized lipoprotein could beprevented by high density lipoproteins that are significantly decreasedin hemodialyzed patients. In addition, minerals like selenium andnutrients such as coenzyme Q10 may minimize free radical generation andthus oxidative stress. Folic acid, vitamin B12 and vitamin B6 may beessential in the prevention of hyperhomocysteinemia that may contributeto the endothelial injury (39) and to oxidation of LDL (40) in thesepatients. A diet rich in mono-unsaturated fatty acids (oleic acid,resistant to oxidation) reduced the extent of endothelial injury indiabetes patients (41). Thus it is possible that dietary orpharmacological means may reduce OxLDL and aldehyde-modified LDL and vonWillebrand factor in chronic renal failure and alleviate the enhancedgeneralized atherosclerosis in such patients.

After adjustment for the extent of renal failure, multiple regressionanalysis revealed that both LDL cholesterol levels and endothelialinjury strongly contributed to the variations in OxLDL andaldehyde-modified LDL levels in severe chronic renal failure patients.

Hemodialysis results in platelet and leukocyte activation (42, 43),which generates oxygen radicals and aldehydes that may also contributeto oxidation of LDL. OxLDL and aldehyde-modified LDL may then contributeto thrombogenesis and atherogenesis by stimulating platelets (44).Because of the rather limited number of patients, subgroup analysis tofurther study the interaction between hemodialysis, oxidation of LDL andischemic atherosclerotic disease could not be performed (45).

4. Legend to FIGS. 6 and 7

FIG. 6. Correlation between plasma levels of OxLDL and aldehyde-modifiedLDL (log values) and titers of autoantibodies (log values): regressionline for severe chronic renal failure patients, either on conservativetreatment (▴; - - - ) (r=0.44; p=0.047) or on hemodialysis (▪;

) (r=0.37; p=0.0055). No significant correlation was observed incontrols and in mild chronic renal failure patients.

FIG. 7. Correlation between plasma levels of OxLDL and aldehyde-modifiedLDL (log values) and of von Willebrand factor antigen (log values):regression line for mild chronic renal failure patients (●; -.-.- )(r=0.59; p=0.0057) or for severe chronic renal failure patients eitheron conservative treatment (▴; - - - ) (r=0.69; p=0.0006) or onhemodialysis (▪;

) (r=0.62; p<0.00001). No significant correlation was observed incontrols.

Example 8

Preparation of Reference-Standard for Use in Immunological Assays

1. Introduction

According to the invention it has been found that LDL that is modifiedby treatment with malondialdehyde (MDA) is highly stable. Furthermore,the extent of modification is highly reproducible. LDL modified with MDAin a particular ratio has an identical number of substituted lysines andcan therefore be used as a reference sample in immunological assays.This example shows the preparation of the standard.

2. Material and Methods

MDA-modified LDL was added to control plasma (containing anti-oxidantsand anti-platelet compounds and anti-coagulants) to a finalconcentration of 100 nM MDA modified apo B-100. Aliquots were frozen at−80° C. In 6 days were aliquots were thawed, diluted to finalconcentrations ranging from 10 to 0.1 nM MDA-modified apo B-100 andanalyzed in ELISA (4 dilution curves per day).

3. Results

The inter-assay variation coefficients of 10 subsequent sandwich ELISA'sof this invention using 10 subsequent, independent MDA-modified LDLstandard preparations of this invention are summarized in Table 11.

These data show that for concentrations of MDA-modified LDL ranging from10 and 0.01 nM the inter-assay variation ranged from 7.6 to 16.9%.

Abbreviations

-   C₅₀: concentration required to obtain 50% inhibition of antibody    binding-   MDA: malondialdehyde-   HEMO: severe chronic renal failure patients on maintenance    hemodialysis-   MCRF: mild chronic renal failure patients-   SCRF: severe chronic renal failure patients on conservative    treatment

OxLDL: oxidized low density lipoproteins. TABLE 1 Clinical data of hearttransplant patients Heart transplant patients Dilated cardiomyopathyIschemic heart disease Characteristics (n = 47) (n = 60) p-values Age ofrecipient (yr)  54 ± 1.6   55 ± 0.95 *NS Gender of recipient (M/F) 41/6 53/7  *NS Age of donor (yr)  29 ± 1.5  29 ± 1.4 **NS Gender of donor(M/F) 31/16 44/16 *NS Length of follow up (mo)  39 ± 3.1  50 ± 2.7**0.008 Duration of ischemia (min) 130 ± 7.0  140 ± 5.3  **NS No of HLAmismatches DR  1.5 ± 0.09  1.4 ± 0.08 **NS B + DR  3.1 ± 0.13  3.0 ±0.13 **NS No of rejection episodes 0.38 ± 0.13 0.25 ± 0.06 **NSCytomegalovirus infection 26 43 *NS Hypertension 37 53 *NS Diabetes 4 3*NS Coronary artery disease Grade 0 39 40 *NS Grade I 5 13 *NS Grade II3 7 *NS Lipid lowering drugs 17 39  *0.004 Statins 13 33  *0.006Fibrates 4 6 *NS Calcium channel blockers 31 47 *NSData represent mean ± SEM or number of patients.*p-values determined by Chi-square test.**p-values determined by Dunnett's multiple comparison test.NS: not significant.

TABLE 2 Laboratory data of controls and heart transplant patients Hearttransplant patients Dilated Controls cardiomyopathy (DC) Ischemic heartdisease Characteristics (n = 27) (n = 47) p vs control (n = 60) p scontrol p-valuesvs DC Serum triglyderides (mg/dl)† 130 ± 11  130 ± 8.3 NS 140 ± 7.0  NS NS HDL cholesterol (mg/dl)‡  44 ± 2.1  54 ± 2.5 NS  49± 1.9 NS NS LDL cholesterol (mg/dl)‡ 120 ± 4.7  100 ± 4.4  NS 110 ± 3.3 NS NS Oxidized LDL (mg/dl)  0.59 ± 0.036  1.3 ± 0.14 <0.001  1.7 ± 0.13<0.001 <0.01Data represent mean ± SEM.p-values determined by Dunnett's multiple comparison test.NS: not significant.†to convert values for serum triglycerides to millimoles per liter,multiply by 0.011.‡to convert values for serum cholesterol to millimoles per liter,multiply by 0.026.

TABLE 3 Logistic regression analysis of the relation between clinical-laboratory data and extent of coronary artery stenosis in hearttransplant patients. Independent variable Chi-square value p-valueOxidised LDL 18 0.0001 Length of follow up 11 0.0008 Age of donor 3.90.047 Age of recipient 0.12 0.73 Sex of recipient 1.8 0.18 Sex of donor0.025 0.88 History of pretransplant dilated 0.0018 0.97 cardiomyopathy(n = 47) or ischemic heart disease (n = 60) Duration of ischemia 0.250.62 No of HLA mismatches 1.6 0.20 No of rejection episodes 3.0 0.081Cytomegalovirus infection 0.17 0.47 Hypertension 1.9 0.16 Diabetes0.0016 0.97 Treatment with lipid lowering drugs Statins 1.1 0.30Fibrates 0.12 0.73 Treatment with calcium channel blockers 0.16 0.49Serum triglycerides 0.18 0.67 Serum HDL cholesterol 0.25 0.61 Serum LDLcholesterol 0.044 0.83The data set contained 107 patients.Original cardiac disease was dilated cardiomyopathy in 47 and ischemicheart disease in 60 patients.Coronary artery stenosis was assessed angiographically.All quantitative parameters were transformed logarithmically to obtain anormal distribution for linear regression.Chi-square values were obtained after adjustment for all othervariables.

TABLE 4 Clinical data of heart transplant patients without and withprogression of coronary artery stenosis during a 3 years follow-upperiod. Heart transplant patients Without progression With progressionCharacteristics (n = 34) (n = 12) p-value Age of recipient (yr)  58 ±1.4  60 ± 1.4 **NS Gender of recipient (M/F) 21/14 11/1  *NS Age ofdonor (yr)  25 ± 1.3  32 ± 3.8 **NS Gender of donor (M/F) 27/7  10/2 *NS Duration of ischemia (min) 130 ± 6.7  140 ± 11  **NS No of HLAmismatches DR  1.2 ± 0.13  1.5 ± 0.15 **NS B + DR  2.8 ± 0.21  3.2 ±0.24 **NS Cytomegalovirus infection 24 11 *NS Hypertension 21 10 *NSDiabetes  1  2 *NSData represent mean ± SEM or number of patients.*p-values determined by Chi-square analysis.**p-values determined by Dunnett's multiple comparison test.NS = not significant

TABLE 5 Laboratory data of heart transplant patients without and withprogression of coronary artery stenosis during a 3 years follow-upperiod. Heart transplantation patients Without progression Withprogression Characteristics (n = 34) (n = 12) p-value Serumtriglycerides (mg/dl) 130 ± 8.6  150 ± 14  NS HDL cholesterol (mg/dl) 50 ± 2.7  49 ± 4.9 NS LDL cholesterol (mg/dl) 110 ± 3.6  105 ± 8.7  NSOxidized LDL (mg/dl)  1.2 ± 0.069  2.6 ± 0.33 0.0005Data represent means ± SEM.p-values determined by Dunnett's multiple regression comparison test.NS = not significant.

TABLE 6 Mild chronic Severe chronic renal failure Controls renal failurenon-dialysed hemodialysed Characteristics (n = 27) (n = 20) (n = 21) (n= 56) Males/females 12/15 11/9 7/14 33/23 Age (years) 54 (50-58)* 52(44-60)*  55 (49-62)* 61 (58-65)* Body weight (kg) 72 (69-76)* 73(67-79)*  59 (53-65)* 65 (61-68)* Creatinine clearance  110 (110-120)*34 (29-39)* 8.4 (7-10)*  nd (ml/min) Primary renal disease:Glomerulonephritis — 4 3 11 Autosomal dominant polycystic — 2 6 10kidney disease Diabetes — 1 4 6 Reflux-nephropathy — 1 2 2 ChronicInterstitial Nephritis — 2 2 9 Hypertensive nephropathy — 2 0 2 Other¹ —6 1 9 Unknown — 2 3 7 Hypertension 1 16 19 18 Atherosclerotic heartdisease — 6 7 24 Cerebrovascular accidents — 0 3 9 Peripheral vasculardisease — 2 1 13*Data represent means and 95% confidence intervals (between brackets).¹including: hereditary nephropathy, sarcoidosis, renal tuberculosis,thrombotic thrombocytopenic purpura, myeloma, traumatic loss, congenitalurinary tract abnormalities.nd: creatinine clearance rate cannot be determined adequately.

TABLE 7 Laboratory data of study subjects Controls MCRF patients SCRFpatients HEMO patients (n = 27) (n = 20) p vs controls (n = 21) p vscontrols (n = 56) p vs controls Triglycerides (mg/dl)  120 (100-150) 150(130-170) NS 120 (100-140) NS 130 (110-160) NS HDL cholesterol (mg/dl)  44 (39-48)  38 (33-42) NS  44 (38-50) NS  37 (35-40) <0.05  LDLcholesterol (mg/dl)  120 (110-130) 110 (100-130) NS 110 (105-130) NS 120(110-130) NS Oxidized LDL (mg/dl) 0.59 (0.52-0.66)  1.6 (1.0-2.2) <0.01 1.8 (1.3-2.3) <0.001  3.2 (2.7-3.7) <0.001 Autoantibodies (titer)  4.2(4.0-4.4)  4.7 (4.0-5.4) NS  5.0 (4.2-5.8) NS  6.6 (5.7-7.4) <0.001 vWF(percent)  100 (90-110) 150 (110-180) NS 160 (130-190) <0.01  210(180-240) <0.001Data represent means and 95% confidence intervals (between brackets).

TABLE 8 Characteristics of native LDL and of OxLDL isolated from plasmaof severe chronic renal failure patients Native LDL OxLDLProtein/antigen ratio >100 1.1 Reactivity with mAb-4E6 (C₅₀ mg/dl) 250.02 Relative electrophonetic mobility 1 1.7 Malondialdehyde (mole/moleprotein) 3 68 Substituted lysines per apo B-100 5 130Cholesterol/protein ratio 1.8 3.3 Free cholesterol/cholesterol esterratio 0.38 0.36 Phospholipid/protein ratio 1.7 1.6 Fatty acids (%) 16:014 37 18:1 19 50 18:2 55 10 20:4 12 3Data represent means of ten LDL preparations of chronic renal failurepatients.Native LDL and OxLDL patients were separated by ion-exchangechromatography.

TABLE 9 Multiple regression analysis of the dependence of OxLDL on theextent of renal failure Variable F-value p-value Age 1.2 0.28 Sex 1.40.25 Hypertension 1.1 0.31 Triglycerides 1.5 0.23 HDL cholesterol 1.70.20 LDL cholesterol 0.99 0.32 Renal failure 14 0.0004The data set contained 27 controls, 20 MCRF patients and 21 SCRFpatients.F-values were obtained after adjustment for the other variables.Cockcroft creatinine clearance rates were used as a quantitativeparameter for the extent of renal failure.All linear variables were logarithmically transformed to obtainnormality for linear regression analysis.The multiple R² value of the multiple regression model was 0.634.

TABLE 10 Multiple regression analysis of the dependence of OxLDL onhemodialysis and LDL cholesterol levels in severe chronic renal failurepatients Variable F-value p-value Age 0.11 0.58 Sex 0.19 0.66Hypertension 0.01 0.95 HDL cholesterol 0.02 0.89 Triglycerides 3.7 0.060Hemodialysis 5.6 0.021 LDL cholesterol 7.1 0.0095The data set contained 21 SCRF and 56 HEMO patients.All linear variables were transformed logarithmically to obtainnormality for linear regression analysis.The multiple R² value of the multiple regression model was 0.56.

TABLE 11 Inter-assay variation coefficients of sandwich ELISA using 10subsequent, independent MDA-modified LDL preparations Inter-assayvariations Concentration coefficients (nM) (%) 10 9.6 5 7.6 2.5 8.4 1.2513.2 0.62 12.0 0.31 13.0 0.16 12.3 0.08 15.5 0.04 16.9 0.02 13.6 0.0111.4

DOCUMENTS

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56. An immunological assay for the detection and/or quantification ofhuman MDA-modified LDL (malondialdehyde-modified low densitylipoprotein) and human OxLDL (oxidized low density lipoprotein) in asample derived from the body fluids or tissues of a human being, saidassay comprising: (a) contacting the sample with a first antibody thathas high affinity for human MDA-modified LDL and human OxLDL wherein thefirst antibody binds to the same epitope as monoclonal antibody mAb-4E6produced by hybridoma Hyb4E6 deposited at the BCCM (Belgian CoordinatedCollections of Microorganisms) under deposit accession number LMBP 1660CB on Apr. 24, 1997; and (b) thereafter visualizing and/or quantifying abinding reaction between the first antibody and the MDA-modified LDL andOxLDL present in the sample; wherein the MDA-modified LDL and OxLDL forwhich the first antibody has high affinity contain at least 60substituted lysine moieties per apo B-100 (apolipoprotein B-100) moiety.57. The assay of claim 56 in which the MDA-modified LDL and OxLDL forwhich the first antibody has high affinity contain at least about 90substituted lysine moieties per apo B-100 moiety.
 58. The assay of claim56 in which the MDA-modified LDL and OxLDL for which the first antibodyhas high affinity contain at least about 120 substituted lysine moietiesper apo B-100 moiety.
 59. The assay of claim 56 in which theMDA-modified LDL and OxLDL for which the first antibody has highaffinity contain at least about 210 substituted lysine moieties per apoB-100 moiety.
 60. The assay of claim 56 in which the MDA-modified LDLand OxLDL for which the first antibody has high affinity contain atleast about 240 substituted lysine moieties per apo B-100 moiety. 61.The assay of claim 56 which is a competitive assay.
 62. The competitiveassay of claim 61 in which MDA-modified LDL and/or OxLDL are bound to asubstrate, comprising contacting the sample and the first antibody withthe substrate having bound to it MDA-modified LDL and OxLDL.
 63. Theassay of claim 56 which is a sandwich assay in which the first antibodyis bound to a substrate, comprising contacting the sample with thesubstrate having bound to it the first antibody.
 64. The assay of claim63 in which a second antibody is used and the second antibody has highaffinity for human MDA-modified LDL and human OxLDL.
 65. The assay ofclaim 64 in which the second antibody has high affinity for human nativeLDL (low density lipoprotein).
 66. The assay of claim 56 which is animmunohistochemical assay in which the sample is a tissue sample and itis contacted with the first antibody.
 67. The assay of claim 56 in whichthe affinity constant of the first antibody for MDA-modified LDL and forOxLDL is at least about 1×10¹⁰ M⁻¹.
 68. The assay of claim 56 in whichthe first antibody has low affinity for human native LDL.
 69. The assayof claim 68 in which the affinity constant of the first antibody forhuman native LDL is less than about 1×10⁶ M⁻¹.
 70. The assay of claim 64in which the affinity constant of the second antibody for MDA-modifiedLDL and for OxLDL is at least about 1×10¹⁰ M⁻¹.
 71. The assay of claim70 in which the affinity constant of the second antibody for humannative LDL is at least about 1×10⁹ M⁻¹.
 72. The assay of claim 71 inwhich the second antibody is the monoclonal antibody mAb-8A2 produced byhybridoma Hyb8A2 deposited at the BCCM under deposit accession numberLMBP 1661 CB on Apr. 24,
 1997. 73. The assay of claim 56 in which thesample is derived from the body fluids of a human being.
 74. The assayof claim 56 in which at least one antibody is capable of detecting 0.02mg/dl of human MDA-modified LDL and human OxLDL in undiluted humanplasma.